EP0252525A2 - Verfahren zur Bestimmung von alpha-Amylase unter Anwendung von modifizierten Oligosacchariden und Verfahren zu deren Herstellung - Google Patents

Verfahren zur Bestimmung von alpha-Amylase unter Anwendung von modifizierten Oligosacchariden und Verfahren zu deren Herstellung Download PDF

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EP0252525A2
EP0252525A2 EP87110023A EP87110023A EP0252525A2 EP 0252525 A2 EP0252525 A2 EP 0252525A2 EP 87110023 A EP87110023 A EP 87110023A EP 87110023 A EP87110023 A EP 87110023A EP 0252525 A2 EP0252525 A2 EP 0252525A2
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group
formula
halogen atom
hydrogen
oligosaccharide
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EP0252525A3 (en
EP0252525B1 (de
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Tokuji Ikenaka
Kaoru Omichi
Shinji Satomura
Yuko Natsuka
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Fujifilm Wako Pure Chemical Corp
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Wako Pure Chemical Industries Ltd
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Priority to AT87110023T priority patent/ATE97131T1/de
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
    • C08B37/0015—Inclusion compounds, i.e. host-guest compounds, e.g. polyrotaxanes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/16—Preparation of compounds containing saccharide radicals produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/20—Preparation of compounds containing saccharide radicals produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/40—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving amylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
    • C12Q2334/10—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases p-Nitrophenol derivatives
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/12—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar
    • G01N2400/14—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar alpha-D-Glucans, i.e. having alpha 1,n (n=3,4,6) linkages between saccharide units, e.g. pullulan
    • G01N2400/18—Cyclodextrin

Definitions

  • This invention relates to a process for determining the activity of a-amylase in a sample using a modified oligosaccharide as a substrate and a process for producing such a modified oligosaccharide.
  • U.S. Patent No. 4,649,108 to Blair discloses a method of measuring the amount of a-amylase in liquid sample comprising the steps of providing an oligosaccharide substrate for a-amylase, said substrate containing at least 3 glucose units, the reducing-end glucose residue (unit) being bonded, via a bond cleavable by a-or ⁇ -glucosidase, to a label which exhibits an optically measurable change upon cleavage of said bond, and the non-reducing-end (terminal) glucose residue being bonded to a blocking group which inhibits cleavage by exo-enzymes of the bond between said non-reducing-end glucose redidue and the adjacent glucose residue; contacting said sample with said oligosaccharide substrate and with a first added exo-enzyme capable of cleaving
  • the blocking groups can be carboxylic acid esters, phosphate esters, sulfonate esters, ethers (such as benzyl, silyl, and triphenylmethyl), monosaccharides other than a-1,4 linked glucose, and acetal or ketal blocking groups such as benzylidene (column 4 lines 37-67).
  • ester blocking groups are unstable since they are easily hydrolyzed in an aqueous solution. Further, when the size of the blocking group becomes larger as in the case of toluenesulfonyl, methanesulfonyl, silyl or triphenylmethyl, the solubility in water is undesirably lowered.
  • the substrate has eight or fewer glucose units, and most preferably has six or seven, and preferred blocking substituents are acetals or ketals, e.g. benzylidene (column 2 lines 4-7).
  • This invention provides a process for determining the activity of a-amylase in a sample which comprises
  • This invention also provides a process for producing an oligosaccharide having a special substituent at the non-reducing-end glucose residue such as that represented by the formula (I) or (II), which comprises reacting a modified cyclodextrin with cyclomaltodextringluconotransferase in the present of an acceptor, and then reacting with glucoamylase or a-glucosidase.
  • This invention further provides a process for producing an oligosaccharide having a special substituent at the non-reducing-end glucose residue such as that represented by the formula (I), which comprises reacting an oligosaccharide having a substituend which exhibits an optically measurable change upon cleavage at the reducing-end glucose residue with an aldehyde, a ketone, an acetal or a ketal to form 4,6-O-cyclic acetal or 4,6-0-cyclic katal at the non-reducing-end glucose residue, and reducing said 4,6-0- cyclic acetal or ketal.
  • a process for producing an oligosaccharide having a special substituent at the non-reducing-end glucose residue such as that represented by the formula (I), which comprises reacting an oligosaccharide having a substituend which exhibits an optically measurable change upon cleavage at the reducing-end glucose residue with an aldehyde, a ketone, an acetal or a
  • the modified oligosaccharide used as a substrate for a-amylase is represented by the formula: or wherein R 1 is a halogen atom such as bromine, chlorine, iodine, etc.; and R 2 is a group of the formula: in which R 3 through R 6 are independently hydrogen, a lower alkyl group preferably having 1 to 4 carbon atoms, a lower alkoxy group preferably having 1 to 4 carbon atoms, a nitro group, a carboxyl group, a sulfonic acid group, or a halogen atom such as chlorine, bromine, iodine, etc., and R 3 and R 5 , and/or R 4 and R 6 , may be bonded to form an aromatic ring; R 7 is hydrogen, a lower alkoxy group preferably having 1 to 4 carbon atoms, a halogen atom such as chlorine, bromine, iodine, etc., or a nitro group; R 8 is hydrogen, a methyl group,
  • the oligosaccharides of the formula (I) and (II) have advantages in that the hydrolysis rate is fast, that is, the sensitivity as a substrate is high, the Michaelis constant (K m ) which is defined as a substrate concentration for giving a half of the maximum rate (V max ) of a-amylase is small, that is the specificity as a substrate is high and the required substrate concentration for measuring activities of a-amylase is low (usually used in a concentration of the value of K m X 5 to 10), and the position of hydrolysis is almost one position, that is, the color production reaction from the hydrolyzed products can be carried out simply and easily.
  • the hydrolysis rate is measured by using human pancreatic a-amylase (HPA) or human salivary a-amylase (HSA) for each substrate (1.0 mM) at pH 7, and evaluating by means of relative values by taking the value of G5P as 1.
  • HPA human pancreatic a-amylase
  • HSA human salivary a-amylase
  • the K m values were calculated by least square method according to a Lineweaver-Burfe plot. The amounts of the products were measured with HPLC.
  • the binding mode of each substrate were measured with high-performance liquid chromatography (HPLC) as described above.
  • the activity of ⁇ -amylase can be measured by using the modified oligosaccharide of the formula (I) or (II) as follows: G: glucose residue exo-enzyme(s):
  • the substituent R O is attached to the C 6 position of the non-reducing-end glucose residue and the substituent OR 2 is attached to the C 1 position of the reducing end glucose residue and represented by, for example, a phenoxy group which may have one or more substituent groups, a naphthoxy group which may have one or more substituent groups, an umbelliferyl group which may have one or more substituent groups, or an indoxyl group which may have one or more substituent groups.
  • the is the a-amylase in a sample first acts on the modified oligosaccharide of the formula (I) or (II) to produce wherein the primary alcohol group (-CH 2 0H) at the C 6 position of the non-reducing end glucose residue is replaced by the substituent R°, and G-G-OR 2 wherein OR 2 is as defined above. Then, G-G-OR 2 is reacted with coupling enzyme(s) such as glucoamylase, a-glucosidase, ⁇ -glucosidase and athe like to produce 2G and R 2 -OH which exhibits an optically measurable change.
  • coupling enzyme(s) such as glucoamylase, a-glucosidase, ⁇ -glucosidase and athe like to produce 2G and R 2 -OH which exhibits an optically measurable change.
  • R 2 -OH is a nitrophenol such as p-nitrophenol
  • the activity of a-amylase in a sample can be obtained by directly measuring the absorption spectrum thereof (e.g. absorbance at 405 nm).
  • R 2- OH is phenol or naphthol having no nitro group (or capable of having a nitro group) such as phenol, o-chlorophenol, 2,6-dichlorophenol, p-methoxyphenol, or the like
  • a coupler such as 4-aminoantipyrine, or 3-methyl-2-benzothiazolinone hydrazone (MBTH)
  • an oxidase such as catechol oxidase, laccase, tyrosinase or monophenol oxidase, or an oxidant such as iodic acid or periodic acid; or peroxidase and hydrogen peroxide to produce a dye, the absorption spectrum of which is measured.
  • R 2 -OH is a compound emitting fluorescence such as umbelliferone or 4-methylumbelliferone
  • the fluorescence intensity is measured.
  • R 2. OH is indoxyl
  • the absorption spectrum of indigo dyes produced by oxidation is measured.
  • the concentration of the modified oligosaccharide used as a substrate in the determination of the activity of a-amylase is not particularly limited but usually is about 0.1 to 10 mM.
  • any ones containing a-amylase can be used.
  • exo-enzyme which is a coupling enzyme there can be used a-glucosidase, a mixture of ⁇ -glucosidase and glucoamylase, a mixture of a-glucosidase and 0-glucosidase, a mixture of a-glucosidase, ⁇ -glucosidase and glucoamylase, a mixture of a-glucosidase and isomaltase, and a mixture of isomaltase and glucoamylase.
  • exo-enzymes may be derived from animals, plants and microorganisms.
  • the amount of exo-enzymes used is usually 0.5 to 100 units/ml, preferably 2 to 50 units/ml.
  • the reaction temperature is not particularly limited and preferably about 25° to 40°C.
  • the reaction time can be selected properly depending on the purposes.
  • the pH of the reaction is not particularly limited and preferably about 6 to 8.
  • a buffer for maintain the pH proper there can be used a phosphate buffer, a tris(hydroxymethyl)aminomethane-HCI buffer, a Good's buffer, and the like.
  • an activation imparting agent for a-amylase there can be used sodium chloride, calcium chloride, potassium chloride, calcium acetate and the like.
  • the coupler for coupling the phenol or naphthol liberated by the action of the exo-enzyme(s)
  • 4-aminoantipyrine 3-methyl-2-benzothiazolinonehydrazone (MBTH)
  • MBTH 3-methyl-2-benzothiazolinonehydrazone
  • p-amino-N,N-diethylaniline and the like.
  • oxidase for coupling (oxidation condensation) of the phenol or naphthol with the coupler there can be used laccase, catechol oxidase, tyrosinase, monophenol oxidase, and the like, derived from animals, plants, and microorganisms, in an amount of usually 0.2 to 10 units/ml, preferably 0.5 to 4 units/ml.
  • iodic acid and/or a salt thereof such as sodium, potassium, and the like salt
  • periodic acid and/or a salt thereof such as sodium, potassium salt, hydrogen peroxide, and the like.
  • the modified oligosaccharide of the formula (I) or (II) cannot be used as a substrate for glucoamylase, a-glucosidase, ⁇ -glucosidase or isomaltase as it is. But since the modified oligosaccharide of the formula (I) or (II) is easily soluble in water and excellent in affinity for a-amylase, it can be a good specific substrate for a-amylase.
  • the a-amylase determination process using as a substrate the modified oligosaccharide of the formula (I) or (II) does not bring about side reactions and has a very small blank value. Further the reagent solution for the measurement is very stable. In addition, since human a-amylase is almost hydrolyzed one glycosidic linkage of the modified oligosaccharide of formula (I) or (II), the stoichiometry is established, which results in making the kinetic determination of a-amylase possible.
  • the duration of the lag period can be shortened by reacting with at least one coupling enzyme (exo-enzyme) such as glucoamylase, a-glucosidase, isomaltase or ⁇ -glucosidase in the reaction after the a-amylase reaction.
  • exo-enzyme such as glucoamylase, a-glucosidase, isomaltase or ⁇ -glucosidase in the reaction after the a-amylase reaction.
  • the measurement of an optically measurable change can be carried out by measuring absorption spectra of nitrophenols or indigo dyes liberated, by measuring absorption spectra of dyes formed by oxidation coupling of phenols or naphthols liberated with 4-aminoantipyrine, MBTH, or the like, or by measuring fluorescence intensity of umbelliferone liberated, so that the determination process is hardly influenced by sugars such as glucose, maltose and the like, reducing substances such as ascorbic acid, bilirubin, and the like present in samples to be measured.
  • the a-amylase activity determination process of this invention can be applied to either a rate assay wherein the reaction rate under certain conditions is measured, or an end point assay wherein a reaction terminator is used.
  • a-amylase activity determinatin process of this invention can well be suited to an autoanalyzer and if necessary can be applied to a manual method.
  • the process of this invention can also be applied to a test paper method which is a very simple method and a so-called dry determination process wherein multi-layer analysis sheets containing reaction reagents (multi-layer one- body type quantitative analysis films) are used.
  • the modified oligosaccharides of the formula (I) and (II) can be produced by reacting a modified cyclodextrin with cyclomaltodextrin-glucanotransferase in the presence of an acceptor, and then reacting with glucoamylase or a-glucosidase.
  • Such a process can also be applied to the production of a variety of compounds similar to the oligosaccharides of the formula (I) and (II).
  • R O is an alkoxymethyl group, a benzyloxymethyl group, an aminomethyl group, a carboxyl group or a halomethyl group
  • R 2 is the same group as defined in R 2 or a glucitol residue
  • n is an integer of 2 to 5.
  • modified cyclodextrin there can preferably be used those having one modified glucose residue per molecule, and may contain those having two or more modified glucose residues per molecule.
  • substituted glucose wherein the hydroxyl group in the glucose is replaced by a substituent which can emit fluorescence, e.g. a 2-pyridylamino group, 3-pyridylamino group, etc.; a substituent which can absorb UV light, e.g.
  • an anilino group an anilino group, a methylanilino group, a hydroxyanilino group, a carboxyphenylamino group, etc; an alkoxy group such as a methoxy group, an ethoxy group, etc.; a substituted alkoxy group such as a carboxy-methoxy group, a hydrox- yethoxy group, a benzyloxy group, a phenetyloxy group, a pyridylmethyloxy group, etc.; a halogen atom such as chlorine, bromine, etc.; a hydrazono group, a phenylhydrazono group or an amino group; or there can be used glucuronic acid wherein the -CH 2 0H group at the C 6 position of glucose is replaced by a -COOH group.
  • the acceptor there can be used glucose, maltose, maltotriose, and derivatives thereof having as a substituent, e.g. p-nitrophenyl, phenyl, umbelliferyl, naphtyl,
  • a substituent e.g. p-nitrophenyl, phenyl, umbelliferyl, naphtyl
  • the glucose chain becomes longer, the glucose chain of major product in the initial reaction also becomes longer. Therefore, the glucose chain length of 1 to 3 is properly selected and used as the acceptor depending on the glucose chain length of the desired product.
  • the position of substituent is preferably the C 1 position, irrespective of a-substitution or ⁇ -substitution.
  • Glucose derivatives having one or more substituents at C 2 , C 3 and/or C 6 positions are not so preferable since the reaction is difficult to proceed.
  • the substituents at C 2 , C 3 and/or C 6 positions it is preferable to use maltase wherein one or more substituents are introduced into C 2 , C 3 and/or C 6 positions of the reducing end glucose residue.
  • the -OR 2 o groups are a p-nitrophenoxy group, a m-nitrophenoxy group, an o-chlorophenoxy group, a p-chlorophenoxy group, a 2,6-dichlorophenoxy group, an o-methoxyphenoxy group, a p-methoxyphenoxy group, an o-methylphenoxy group, an o-carboxyphenoxy group, an o-sulfophenoxy group, a 1-naphthoxy group, a 2-sulfo-1-naphthoxy group, a 2-carboxy-1-naphthoxy group, an umbelliferyl group, a 4-methylumbelliferyl group, an indoxyl group, glucitol residue, etc.
  • CCTase The cyclomaltodextrin-glucanotransferase (hereinafter referred to as "CGTase”) is not particularly limited to its origin and source. There can be used those derived from Bacillus macerans, Bacillus megaterium, Klebsiella pneumonie, alcaligenous, etc.
  • the modified cyclodextrin can easily be obtained by using a-, ⁇ -or ⁇ -cyclodextrin as a starting material and reacting with various reactants for introducing the desired modifying groups thereinto according to processes for producing various modified glucoses described in, for example, Method in Carbohydrate Chemistry I (1962) to V (1965), published by Academic Press.
  • the selection of a-, ⁇ -or -y-cyclodextrin is optinal and can be determined depending on the glucose chain length of the desired modified oligosaccharide derivative and its maximum yield.
  • the pH of the reaction of the modified cyclodextrin with CGTase in the presence of an accptor changes slightly depending on the origin of CGTase, but usually 6 to 8.
  • Any buffering agent which does not inhibit the enzymatic reaction can be used for maintaining the suitable pH.
  • the buffering agents are Good's buffer, ammonium acetate buffer, carbonate buffer, phosphate buffer, etc.
  • the modified cyclodextrin is used in a concentration of preferably 1 to 50 mmol/I and the accpetor is used in a concentraiton of 1 mmol/I or more to the solubility limit.
  • the acceptor is preferably used in an amount of 5 moles or more per mole of the modified cyclodextrin.
  • the CGTase is preferably used in an amount of 50 to 5000 U/ml.
  • the reaction is preferably carried out at 20 to 50°C.
  • CGTase is deactivated by heating, for example at 90°C or higher for 10 minutes or more, or by changing the pH, for example pH of 4.0 or less.
  • the reaction using glucoamylase or a-glucosidase can preferably be carried out at most suitable pH, for example pH 4 to 6, and using glucoamylase or c-glucosidase in an amount of preferably 5 to 100 U/ml.
  • the modified oligosaccharide of the formula (I) can be produced by reacting an oligosaccharide having a substituent which exhibits and optically measurable change upon cleavage at the reducing-end glucose residue with an aldehyde, a ketone, an acetal or a ketal to form 4,6-0-cydic acetal or 4,6-O-cyclic ketal at the non-reducing end glucose residue, and reducing said 4,6-0-cyclic acetal or ketal.
  • Such a process can also be applied to the production of a variety of compounds similar to the oligosaccharide of the formula (I).
  • R is a benzyl group, a substituted benzyl group (the substituent group is a lower alkyl group preferably having 1 to 4 carbon atoms, a lower alkoxy group preferably having 1 to 4 carbon atoms, an alkyl-substituted amino group, a carboxyl group, a nitro group, or a halogen atom such as chlorine, bromine, iodine, etc.), a 2-, 3-or 4-pyridylmethyl group, a straight-or branched-chain or cyclic alkyl group having 1 to 6 carbon atoms, or an alkenyl group having 1 to 6 carbon atoms; R 2 0 is the same group as defined in R 2 or a glucitol residue; and n is an integer of 2 to 5.
  • the oligosaccharide used as a starting material can be represented by the formula: wherein R 2 0 and n are as defined above.
  • the oligosaccharide derivative of the formula (VIII) is reacted with an aldehyde, a ketone, an acetal or a ketal to form a cyclic acetal between the C 4 and C 6 positions of the non-reducing-end-glucose residue.
  • the oligosaccharide derivative of the formula (VIII) is available commercially or can be synthesized by a process described in, for example, Japanese Patent Unexamined Publication No. 54-51892.
  • aldehyde there can be used aromatic aldehyde such as benzaldehyde, tolylaldehyde, etc.; aliphatic aldehydes such as formaldehyde, acetaldehyde, propionaldehyde, butylaldehyde, etc.
  • ketone there can be used acetone, methyl ethyl ketone, etc.
  • acetal there can be used methylal, benzaldehyde dimethylacetal, acetaldehyde dimethylacetal, acetaldehyde diethylacetal, etc.
  • ketal there can be used methyl ethyl ketone ethyleneketal, cyclohexanone dimethylketal, etc.
  • any aldehydes, ketones, acetals and ketals which can form a cyclic acetar or cyclic ketal between the C 4 and C 6 positions of the non-reducing end glucose residue of the oligosaccharide derivative of the formula (VII).
  • the aldehyde, ketone, acetal or ketal is used in an amount of preferably 1 to 100 moles per mole of the oligosaccharide derivative (VIII).
  • the reaction is preferably carried out in the presence of a Lewis acid catalyst such as p-toluenesulfonic acid, zinc chloride, or the like, in a suitable solvent such as N,N-dimethylformamide (DMF), and the like at from room temperature to a reflux temperature for 0.5 to 12 hours.
  • a Lewis acid catalyst such as p-toluenesulfonic acid, zinc chloride, or the like
  • a suitable solvent such as N,N-dimethylformamide (DMF), and the like
  • the resulting intermediate having the cyclic acetal is usually subjected to modification of other hydroxyl groups by acylation, followed by a reduction step.
  • the acylation can be carried out by a conventional process, for example, in the presence of a base such as pyridine and using as an acylating agent such as acetic anhydride, acetylchloride, benzoylchloride, etc.
  • a base such as pyridine
  • an acylating agent such as acetic anhydride, acetylchloride, benzoylchloride, etc.
  • the acylation step is not essential but is preferable to enhance the solubility in a reaction solvent used in the next step (the reduction step).
  • the reduction step is usually carried out by using a reducing agent in the presence of an acid catalyst such as aluminum chloride, boron trifluoride, zinc chloride, p-toluenesulfonic acid, methanesulfonic acid, hydrogen chloride gas (or blown into ether) etc., in a suitable solvent (e.g. tetrahydrofuran (THF), etc.) usually at 0 - 50°C for several tens minutes to several hours.
  • an acid catalyst such as aluminum chloride, boron trifluoride, zinc chloride, p-toluenesulfonic acid, methanesulfonic acid, hydrogen chloride gas (or blown into ether) etc.
  • a suitable solvent e.g. tetrahydrofuran (THF), etc.
  • the reducing agent there can be used sodium cyanoborohydride, lithium aluminumhydride, pyridineborane, dimethylamineborane, trimethylamineborane, t-butylamineborane, diborane, etc.
  • the reud- cing agent is preferably used in an amount of 1 to 1000 moles per mole of the oligosaccharide derivative (VIII).
  • the acid catalyst is preferably used in an amount of 0.5 to 5 moles per mole of the reducing agent.
  • deacylation is conducted by a conventional process, for example, by treating with a 0.01 to 1.0 N sodium methoxidemethanol solution for several to several tens hours at from room temperature to slightly elevated temperatures to easily give the modified oligosaccharide of the formula (VII).
  • Individual after-treatments after individual steps can be carried out by conventional processes.
  • the final product can be purified by a conventional process such as column chromatography, and the like.
  • the lower alkyl group in the substituted benzyl group includes a methyl group, an ethyl group, a propyl group, a butyl group, etc.;
  • the lower alkoxy group in the substituted benzyl group includes a methoxy group, an ethoxy group, a propoxy group, a butoxy group, etc.;
  • the alkyl-substituted amino group includes a dimethylamino group, a diethylamino group, an N-ethyl-N-( ⁇ -hydroxyethyl)amino group;
  • the straight-or branched-chain or cyclic clkyl group having 1 to 6 carbon atoms includes a methyl group, an ethyl group, an iso-propyl group, a n-butyl group, a tert-butyl group, a n-pentyl group, an iso-amyl group, a n-hexy
  • the group of the formula: in the formula (VII) is bonded to the reducing end glucose residue and can be hydrolyzed by the action of glucoamylase[E.C.3.2.1.3], «-glucosidase[E.C.3.2.1.20.], ⁇ -glucosidase [E.C.3.2.1.21.], isomaltase-[E.C.3.2.1.10] or ⁇ -amylase[E.C.3.2.1.2.], etc., to form nitrophenols which have absorptions by themselves at visible light range, to finally form dyes by coupling with couplers by the action of oxidases such as catechol oxidase, laccase, tyrosinase and monophenol oxidase, or to finally form dyes by coupling with couplers by the action of oxidants.
  • oxidases such as catechol oxidase, laccase, tyrosinase and monophenol oxidase
  • Examples of the group of the formula:-OR 2 0 in the formula (VII) are a p-nitrophenoxy group, a m-nitrophenoxy group, an o-chlorophenoxy group, a p-chlorophenoxy group, a 2,6-dichlorophenoxy group, a 2-chloro-4-nitrophenoxy group, an o-methoxyphenoxy group, a p-methoxyphenoxy group, an o-methylphenoxy group, an carboxyphenoxy group, an o-sulfophenoxy group, a 1-naphthoxy group, a 2-sulfo-1-naphthoxy group, a 2-carboxy-1-napthoxy group, etc.; an umbelliferyl group, a 4-methylumbelliferyl group, a 4-trifluoromethylumbelliferyl group, etc.; an indoxyl group, 5-bromoindoxyl group, 4-chloro-3-bromoindoxyl group, etc; a g
  • the compounds of the formulae (VI) and (VII) can be used as substrates for determining the activity of a-amylase.
  • a part of the modified oligosaccharide of this inventions can effectively be used as a substrate for a fractional measuring method of a-amylase derived from the salivary glands and a-amylase derived from pancreas, that is, a fractional measuring method of a-amylae isozymes wherein decomposed products produced by hydrolysis of a-amylase are reacted with two or more coupling enzymes having different substrate specificities and the produced products are measured to conduct fractional measurements of ⁇ -amylase derived from human pancreas and ⁇ -amylase derived from human salivary glands.
  • the structure was identified by the method disclosed in Japanese Patent Unexamined Publication No. 61-83195.
  • CMG5P p-nitrophenyl O-(6-O-carboxymethyl)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-O- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-O- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-O- ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ -D-glucopyranoside
  • CM- ⁇ -CD mono-O-carboxymethyl- ⁇ -cyclodextrin
  • the number of glucose residue per phenyl group in the resulting carboxyl-G5P. was analyzed by gas-liquid chromatography (GLC) as follows.
  • Glucose residue and carboxyl glucose residue in carboxyl G5P were analyzed by G.L.C. (column, 2% OV-17 on Chromosorb W. (mfd. by Wako Pure Chemical Industries, Ltd., 0.4 x 200 cm) after methanolysis (1.4 M HCI-methanol, 90°C for 2 hours) followed by trimethylsilylation with hexamethyldisilazane and trimethylsilyl chloride in pyridine.
  • the temperature was programmed from 110 to 250°C at the rate of 4°C increments per minute.
  • the concentration of phenyl group was taken as unity, and the content was estimated from the absorbance at 265 nm in 0.1 M acetic acid solution. Further, the phenyl group was measured quantitatively from the absorbance at 265 nm in 0.1 M acetic acid. As a result, the ratio of glucose/phenol in the carboxyl-G5P was 3.9.
  • the number of glucose residue per phenyl group in the amino-G5P was measured by gas-liquid chromatography in the same manner as described in Example 3. As a result, the ratio of glucoseiphenol in amino-G5P was 3.6.
  • amino-G5P was not acted by glucoamylase and the color development by ninhydrin was observed on a TLC plate.
  • HPLC content 96% [column: silica gel Z-ODS, 5C 18 (a trade name, mfd. by Wako Pure Chemical Industries, Ltd., 10 x 300 mm); eluate: linear gradient of 10% CH 3 CN - 0.1% AcOH and 90% CH 3 CN - 0.1% AcOH, flow rate 3 ml/min, measured at 305 nm]
  • G.L.C. analyses ... Glucose residue and benzyl glucose residue in BG5P were analyzed by G.L.C. (column, 2% OV-17 on chromosorb mfd. by Wako Pure Chem. Ind. Ltd., 0.4 x 200 cm) after methanolysis (1.4 M HCI-methanol, 90°C for 2h) followed by trimethylsilylation with hexamethyldisilazane and trimethylsilyl chloride in pyridine. The temperature was programmed from II0 to 250°C at the rate of 4°C increments per minute. The concentration of p-nitrophenyl group was taken as unity, and the content was estimated from the absorbance at 305 nm in 0.1 M acetic acid solution.
  • HPLC content 93% (measuring conditions were the same as described in Example 6).
  • the ratio of glucose/p-nitrophenol of MG5P obtained in the same manner as described in Example 6 was 3.9.
  • the resulting syrup was transferred to a I-liter four-necked flask and dissolved in 250 ml of THF, followed by addition and dissolution of 3 g of dimethylamine borane.
  • 10 ml of a solution of 0.5 M HCI (gas) in diethyl ether was added dropwise with stirring, and the reaction solution was poured into 200 ml of saturated sodium hydrogen carbonate to stop the reaction, followed by extraction with 200 ml of chloroform. After washing twice with saturated saline solution, the solvent was removed in vacuo.
  • 500 ml of methanol solution containing O.IN sodium methoxide was added and stirred at 25°C for 4 hours, followed by addition of acetic acid for neutralization.
  • HPLC content 95% (measuring conditions were the same as described in Example 6)
  • the resulting syrup was transferred to a I-liter four-necked flask and dissolved in 250 ml of THF, followed by addition and dissolution of 3 g of dimethylamine borane.
  • 10 ml of a 0.5 M HCI (gas) in diethyl ether was added dropwise with stirring.
  • the reaction solution was poured into 300 ml of a solution of saturated sodium hydrogen carbonate to stop the reaction, followed by extraction with 200 ml of chloroform. After washing with saturated saline solution twice and removing the solvent in vacuo, 500 ml of a methanol solution containing O.IN sodium methoxide was added thereto, followed by stirring at 25°C for 4 hours. Then, acetic acid was added for neutralization.
  • the resulting syrup was transferred to a I-liter four-necked flask and dissolved in 250 ml of THF, followed by addition and disslution of 3 g of dimethylamine borane.
  • 10 ml of a 0.5 M HCI (gas) in diethyl ether was added dropwise with stirring.
  • the resulting syrup was transferred to a I-liter four-necked flask and dissolved in 250 ml of THF, followed by addition and dissolution of 3 g of dimethylamine borane.
  • 10 ml of a 0.5 M HCI (gas) in diethyl ether was added dropwise with stirring.
  • a measuring reagent solution was prepared by dissolving 30 mg of BG5P obtained in Example 6 in 30 ml of 50 mmol/I 2-(N-morpholino)ethanesulfonic acid (MES)-NaOH buffer (pH 6.9) containing 400 units of glucoamylase, 300 units of a-glucosidase and 20 mmol/I of calcium chloride.
  • MES 2-(N-morpholino)ethanesulfonic acid
  • a-amylase activity of a sample serum was obtained.
  • Fig. 6 shows a relationship between the ⁇ -amylase activity at individual diluted stages of a standard sample (Somogyi unit/dl) and the increase in absorbance (AA) per minute at a wavelength of 405 nm.
  • a measuring reagent solution was prepared by dissolving 2.1 g of N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 230 mg of NaCl, 58 mg of CaC1 2 , 500 mg of NaOH in distilled water, making the total amount 100 ml (pH 7.3) and dissolving 125 mg of BG5P obtained in Example 9 therein. Solution
  • a measuring reagent solution was prepared by dissolving 30 mg of BrG5P obtained in Example 8 in 30 ml of 50 mmol/I MES-NaOH buffer (pH 6.9) containing 400 units of glucoamylase, 300 units of a-glucosidase and 20 mmol/I of calcium chloride.
  • Fig. 7 shows a relationship between the a-amylase activity at individual diluted stages of a standard sample (Somogyi unit/dl) and the increase in absorbance (AA) per minute at a wavelength of 405 nm.

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EP87110023A 1986-07-11 1987-07-10 Verfahren zur Bestimmung von alpha-Amylase unter Anwendung von modifizierten Oligosacchariden und Verfahren zu deren Herstellung Expired - Lifetime EP0252525B1 (de)

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JP62002730A JPH0630602B2 (ja) 1986-07-11 1987-01-09 非還元末端修飾オリゴサッカライド誘導体の新規な製造法
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0324912A2 (de) * 1987-11-20 1989-07-26 Miles Inc. Schnelles Verfahren zur Herstellung von chromogenen Verbindungen, die als Substrate für alpha-Amylase wirken, mit hohen präparativen Ausbeuten
EP0379619A1 (de) * 1987-07-30 1990-08-01 Oriental Yeast Co., Ltd. Verfahren zur selektiven Bestimmung von Alpha-Amylase-Isozymen
EP0416514A2 (de) * 1989-09-04 1991-03-13 Roche Diagnostics GmbH Verfahren zur spezifischen Bestimmung von Pankreas-alpha-Amylase
WO1992007947A1 (en) * 1990-11-01 1992-05-14 Oy Alko Ab Oligosaccharide mixture, and procedure for its after-treatment
EP0557021A2 (de) * 1992-02-14 1993-08-25 Wako Pure Chemical Industries Ltd Oligosaccharidderivate für die Alpha-Amylasebestimmung
US5302514A (en) * 1991-06-26 1994-04-12 Kikkoman Corporation Maltooligoside derivative, reagent for determining α-amylase activity containing the same as effective ingredient and a process for determining the α-amylase activity using same

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* Cited by examiner, † Cited by third party
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CN101831011B (zh) * 2010-05-25 2012-07-18 李晓辉 一种作为药物传输载体的pH响应性环糊精衍生物及其合成方法
CN110747245B (zh) * 2019-11-29 2021-07-27 江南大学 一种利用复合酶制备麦芽低聚糖浆的方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0171960A1 (de) * 1984-07-26 1986-02-19 Genzyme Corporation Verfahren zur alpha-Amylasebestimmung und Reagenssatz
EP0173255A2 (de) * 1984-08-24 1986-03-05 Wako Pure Chemical Industries, Ltd. Oligosaccharidderivate und ihre Verwendung als Substrat um die alpha-Amylase-Aktivität zu messen

Family Cites Families (1)

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Publication number Priority date Publication date Assignee Title
US4649109A (en) * 1984-02-16 1987-03-10 Brandeis University Methods for isolating mutant microorganisms from parental populations

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0171960A1 (de) * 1984-07-26 1986-02-19 Genzyme Corporation Verfahren zur alpha-Amylasebestimmung und Reagenssatz
EP0173255A2 (de) * 1984-08-24 1986-03-05 Wako Pure Chemical Industries, Ltd. Oligosaccharidderivate und ihre Verwendung als Substrat um die alpha-Amylase-Aktivität zu messen

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Vol. 103, No. 1, July 8, 1985, page 256, Abstract 2743P, Columbus, Ohio, US, K. OMICHI et al.: "Preparation of non-reducing-end substituted p-nitrophenyl alpha-maltopentaoside (FG5P) as a substrate for a coupled enzymic assay for alpha-amylases", & J. Biochem. (Tokyo), 1985, 97(4), 977-82 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0379619A1 (de) * 1987-07-30 1990-08-01 Oriental Yeast Co., Ltd. Verfahren zur selektiven Bestimmung von Alpha-Amylase-Isozymen
EP0324912A2 (de) * 1987-11-20 1989-07-26 Miles Inc. Schnelles Verfahren zur Herstellung von chromogenen Verbindungen, die als Substrate für alpha-Amylase wirken, mit hohen präparativen Ausbeuten
EP0324912A3 (de) * 1987-11-20 1990-02-07 Miles Inc. Schnelles Verfahren zur Herstellung von chromogenen Verbindungen, die als Substrate für alpha-Amylase wirken, mit hohen präparativen Ausbeuten
EP0416514A2 (de) * 1989-09-04 1991-03-13 Roche Diagnostics GmbH Verfahren zur spezifischen Bestimmung von Pankreas-alpha-Amylase
EP0416514A3 (en) * 1989-09-04 1991-06-12 Boehringer Mannheim Gmbh Method for specific determination of pancreatic alpha-amylase
AU637819B2 (en) * 1989-09-04 1993-06-10 Boehringer Mannheim Gmbh Process and reagent for the specific determination of pancreatic alpha-amylase
US5264345A (en) * 1989-09-04 1993-11-23 Boehringer Mannheim Gmbh Process and reagent for the specific determination of pancreatic a-amylase
WO1992007947A1 (en) * 1990-11-01 1992-05-14 Oy Alko Ab Oligosaccharide mixture, and procedure for its after-treatment
US5302514A (en) * 1991-06-26 1994-04-12 Kikkoman Corporation Maltooligoside derivative, reagent for determining α-amylase activity containing the same as effective ingredient and a process for determining the α-amylase activity using same
EP0557021A2 (de) * 1992-02-14 1993-08-25 Wako Pure Chemical Industries Ltd Oligosaccharidderivate für die Alpha-Amylasebestimmung
US5319076A (en) * 1992-02-14 1994-06-07 Wako Pure Chemical Industries, Inc. Oligosaccharide derivatives suitable for α-amylase determination
EP0557021A3 (en) * 1992-02-14 1995-12-20 Wako Pure Chem Ind Ltd Oligosaccharide derivatives suitable for alpha-amylase determination

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